1. Field of the Invention
The present invention relates to the use of recombinant DNA technology for the transformation of hosts such as bacteria or plant cells with genetic sequences for the expression of glutamine synthetase (GS). Genes for both the wild type and GS inhibitor resistant enzymes are disclosed.
2. BRIEF DESCRIPTION OF THE BACKGROUND ART
Genetic and molecular biology methods have been used successfully to address various biochemical questions. They have been used, for example, in selecting for increased catalytic activity or altered inducer binding effects in proteins, by systematic amino acid replacement (Oho, et al., Science, 229: 389, 1985; Garges et al., Cell, 41: 745, 1985; Miller, N. The Operon, Miller et al. Eds., Cold Spring Harbor, 1978). However, in general, these studies have been limited to bacterial proteins. The expression of functionally active eukaryotic proteins in prokaryotes should facilitate the structure-function analysis of eukaryotic proteins by the application of many of these techniques, particularly when genetic complementation is feasible.
A particularly interesting enzyme for protein engineering and structure-function analysis is glutamine synthetase (GS) from plants (Miflin et al., in The Biochemistry of Plants: Amino Acids and Derivatives Miflin Ed., Vol. 5, P. 169, Academic Press, 1980). GS, together with glutamate synthase, carries out assimilation of ammonia resulting from molecular nitrogen fixation, nitrate reduction, and catabolism. This enzyme is an octamer with a cube-like shape and usually is composed of a single polypeptide. The production of GS appears to be under both genetic and allosteric regulation.
Several herbicides function by inhibiting plant glutamine synthetase. A typical example of such compounds is the glutamic acid analogue, phosphinothricin (PPT). Unfortunately, many of these herbicides inhibit glutamine synthetase present in the crop plants as well as in weeds, thereby limiting the use of such compounds as PPT.
Since herbicidal selectivity is quite crucial in any commercially useful herbicide, it would be of great interest to be able to confer resistance in selected plants to such non-selective herbicides as PPT, as well as to other GS inhibitors.
There is some precedent for the existence of glutamine synthetases resistant to other compounds. It is known that methione sulfoximine (MSO), another glutamate analog, is a mixed competitive inhibitor (K.sub.i of 0.16 mM) of pea leaf glutamine synthetase (Leason et al., Phytochemistry, 21: 855, (1982)). Miller et al., (Journal of Biological Chemistry, 256: 11307, 1981) studied the properties of several mutants of Salmonella resistant to MSO. In one instance, a mutation occurred which apparently altered the bacterial glutamine synthetase at the ammonia binding domain to confer MSO resistance. More recently, Young et al., (ibid, 258: 11260, 1983) reported that mouse 3T6 cells grown in the presence of MSO developed resistance to this compound. The MSO resistant cells had mRNA enriched for glutamine synthetase, and the authors suggested that this finding implied an amplification of the gene. See also, Sanders et al., Embo Journal, 3: 65 (1984). Neither the Miller, Young nor Sanders studies reported on plant GS.
PPT-resistant alfalfa cells have recently been reported (Newmark, Nature, 305: 383-384, 1983). However, the resistance was due to amplification of the GS gene rather than a specific structural mutation in the GS enzyme expressed. (Donn et al., Journal of Molecular and Applied Genetics, 2: 621-635, 1984).
It would therefore be desirable to develop a bacterial strain which contains the genetic sequence for plant glutamine synthetase. One application of this strain would be for the production of increased levels of GS for commercial or industrial purposes. Another application would be in the development of structural mutants which produce biochemically functional plant glutamine synthetase resistant to the action of GS inhibitors.
Once such a structural GS mutant was developed, its gene could be transformed into plant cells and into plants to provide GS inhibitor-resistant plant cells and plants.